Prevention of recurrence and metastasis of cancer

ABSTRACT

The invention provides methods of preventing and treating cancer, involving the use of attenuated, replication-com-petent, oncolytic herpes viruses.

FIELD OF THE INVENTION

This invention relates to methods of preventing and treating cancer.

BACKGROUND OF THE INVENTION

The impact of cancer on our society cannot be overstated. Cancer is thesecond leading cause of death in the United States, being surpassed onlyby heart disease. Indeed, 1 in 4 deaths in the United States is causedby cancer (American Cancer Society, Cancer Facts and Figures 2001, NewYork 2001, ACS, Inc.).

A cell becomes cancerous when its normal growth control mechanismsbecome impaired. At first, the uncontrolled growth of cancerous cells isconfined to the tissue in which the cells originated but, over time, thecells can spread, or metastasize, from their site of origin to anotherarea of the body. For example, cancer cells may infiltrate the walls ofblood or lymph vessels, thus entering the circulatory or lymphaticsystems, from which they may lodge in another tissue and seed the growthof secondary, metastatic tumors. It is thought that fewer than 1 in10,000 cells that are shed from a primary tumor actually survive, butthis small portion of surviving cells is sufficient to seed secondarytumors elsewhere in the body.

About 35% of patients that are newly diagnosed with cancer lackmetastases, and these patients can be cured by localized treatment oftheir tumors by, e.g., surgery or radiation. The remaining patientseither already have detectable metastases (about 30%) or haveundetectable metastases that will eventually develop into tumors (about35%). Treatment of these patients often involves a systemic approachsuch as, for example, the administration of chemotherapeutic drugs thatinterfere with the growth of rapidly dividing cells, such as cancercells. The overall five-year relative survival rate of all cancers isonly 60%, which underscores the importance of early detection, enablingtumor treatment (e.g., removal) before metastasis occurs, as well as thedevelopment of therapeutic approaches to treating or, preferably,preventing cancer metastasis.

SUMMARY OF THE INVENTION

The invention provides methods of preventing or treating cancer in asubject, e.g., a human subject. The methods involve surgical resectionof a tumor from the subject, followed by administration of anattenuated, replication-competent, oncolytic herpes virus by, forexample, injection into the site of surgical resection. Alternatively,the virus can be injected into the tumor directly, which may then,optionally, be resected. The invention also includes the use of anattenuated, replication-competent, oncolytic herpes virus (e.g., HSV-1)in the preparation of medicaments for carrying out these methods. Theadministered herpes virus prevents or treats the recurrence of anycancer that may remain at the site of resection, as well as prevents ortreats any cancer that may have metastasized from the site of surgicalresection. The metastasized cancer may be found in the lymphatic system,for example, in a lymph node.

Herpes viruses that can be used in the methods of the invention includeherpes simplex virus-1 (HSV-1)-derived viruses, e.g., NV1023.Optionally, the herpes virus administered according to the methods ofthe invention includes a heterologous nucleic acid molecule encoding atherapeutic product, which can be, for example, a cytotoxin, animmunomodulatory protein, a tumor antigen, an antisense nucleic acidmolecule, or a ribozyme. The methods of the invention can also includethe use of a second (or more) anticancer treatment. For example, themethods can be carried out in conjunction with chemotherapy, biologicaltherapy, radiation therapy, or gene therapy.

The invention provides several advantages. For example, when the virusis administered after surgical removal of gross disease, it has as itstarget only microscopic residual tumor, rather than a large tumorvolume, enabling more concentrated, efficient delivery. Also, as isshown in the experiments described below, the virus has oncolyticactivity when injected directly into tumors. Thus, the methods of theinvention can be used to treat primary tumors, as well as to preventlymphatic metastases. The herpes viruses administered according to themethods of the invention follow the same pathways as metastasizing tumorcells, thus enhancing the likelihood of their reaching those areaswithin the lymphatic system, e.g., lymph nodes, that are at greatestrisk for harboring metastatic disease. An additional advantage of themethods of the invention is that they employ mutant herpes viruses thatreplicate in, and thus destroy, dividing cells, such as; cancer cells,while not affecting other, quiescent cells in the body. The herpesviruses can also be multiply mutated, thus eliminating the possibilityof reversion to wild type. Moreover, if necessary, the replication ofthe herpes viruses can be controlled through the action of antiviraldrugs, such as acyclovir, which block viral replication, thus providinganother important safeguard. An additional advantage of usingreplication-competent viruses is that only a fraction of tumor cellsneed to be infected initially, before the viruses propagate inpermissive cancerous tissue. The invention thus provides targeted, safe,and effective methods for preventing and treating primary site cancerrecurrences, as well as regional lymphatic metastases.

Other features and advantages of the invention will be apparent from thefollowing detailed description, the drawings, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, 1C, and 1D: FIG. 1A is a photograph showing a mouse thathas been injected with blue dye at the base of the posterior auricle.FIG. 1B is a photograph showing that injection of the blue dye resultsin rapid blue color detection in an ipsilateral cervical lymph node. Thenormal lymphatic drainage pattern of the murine auricle leads to theipsilateral cervical lymph nodes. FIGS. 1C and 1D are photographsshowing the development of metastatic disease within these same cervicallymph nodes 2 weeks (1C) and 4 weeks (1D) after implantation of squamouscell carcinoma (SCC) VII tumors into the auricle. Approximately 20% ofmice implanted with SCC VII tumors will demonstrate cervical metastasesupon neck exploration.

FIGS. 2A and 2B are photographs showing that the implantation and growthof auricular SCC VII tumors results in histological evidence ofmetastases to the draining cervical lymph nodes. FIG. 2A is an H&E stainof a cervical lymph node showing SCC VII cells first infiltrating thesubcapsular sinus (100×). FIG. 2B is a higher power view of another H&Estained lymph node section showing metastatic SCC VII cells adjacent tonormal lymphocytes (800×).

FIGS. 3A, 3B, and 3C are photographs showing that virally infected cellsmay be detected histologically in the draining cervical lymph nodesfollowing auricular injections of oncolytic herpes virus. FIG. 3A showsthat NV1023 (2×10⁷ pfu) injected into the left auricle results inscattered lacZ-expressing blue cells detected at 24 hours in theipsilateral cervical lymph nodes. FIG. 3B shows a DAPI-stained nodalsection in which NV1066 (2×10⁷ pfu) injected into the left auricle canbe observed under fluorescence microscopy in the ipsilateral cervicallymph nodes by examination at 24 hours. The DAPI stain is used tovisualize all nuclei. FIG. 3C shows cells from an adjacent cervicallymph node section that have been infected with NV1066, which promotesexpression of the green fluorescent protein.

FIG. 4 is a graph demonstrating the reduction of average auricular tumorvolumes due to intratumoral injections of NV1023. Established auriculartumors 6-8 mm in dimension were treated with three serial intratumoralinjections (days 0, 2, and 4) of NV1023 (2×10⁷ pfu). Average auriculartumor volumes were significantly reduced for the virally treated groupat day 7 compared to the PBS treated group (p<0.0001, t-test).

FIGS. 5A and 5B are photographs showing that metastatic deposits of SCCVII within the cervical lymph nodes are successfully infected by NV1023delivered to the surgical beds of excised auricular tumors. FIG. 5A is aphotograph showing an H&E stained section from excised cervical nodesthat demonstrates complete replacement with metastatic SCC VII cells(400×). FIG. 5B is a photograph showing an adjacent nodal sectionstained with X-gal that demonstrates scattered blue-staining metastaticSCC VII cells, reflecting infection by NV1023 (400×).

FIG. 6 is a graph showing that metastatic tumor volume in the cervicallymph nodes is reduced with NV1023 treatment at the primary site.Auricular tumors were excised and the surgical beds treated with 5×10⁷pfu of NV1023. Average cervical nodal volumes were lower for the virallytreated group compared to the PBS treated group (days 6-15).

FIG. 7 is a graph showing that disease free survival is significantlyimproved with NV1023 treatment (5×10⁷ pfu) of the surgical bed followingresection of auricular SCC VII tumors (p<0.05, log rank test).

DETAILED DESCRIPTION

The invention provides methods of preventing and treating cancer. In themethods of the invention, a tumor is surgically removed from a subjectand the site of the resection is treated with an attenuated, replicationcompetent, oncolytic herpes virus. Alternatively, the virus can beinjected directly into a tumor, which may then, optionally, be resected.As is noted above, such viruses selectively replicate in, and thusdestroy, cancer cells, while leaving non-cancerous cells unharmed. Theadministered herpes virus thus eliminates any microscopic diseaseremaining at the site of resection, thereby preventing recurrence atthat site. The administered herpes virus also enters the lymphaticsystem from the site of the primary tumor in the same manner as anypotentially metastasizing tumor cells, thus enabling the treatment andprevention of metastasis from the primary tumor site. Use of theseviruses in the methods of the invention, as well as experimental resultsshowing the efficacy of these methods, are described further below.

Cancers

Examples of cancers that can be prevented or treated using the methodsof the invention include skin (e.g., squamous cell carcinoma, basal cellcarcinoma, or melanoma), breast, colorectal, prostate, brain and nervoussystem, head and neck, testicular, ovarian, pancreatic, lung, liver(e.g., hepatoma), kidney, bladder, gastrointestinal, bone, endocrinesystem (e.g., thyroid and pituitary tumors), and lymphatic system (e.g.,Hodgkin's and non-Hodgkin's lymphomas) cancers. Cancers of thenervous-system include, for example, astrocytoma, oligodendroglioma,meningioma, neurofibroma, glioblastoma, ependymoma, Schwannoma,neurofibrosarcoma, neuroblastoma, and medulloblastoma. Other types ofcancers that can be treated using the methods of the invention includefibrosarcoma, neuroectodermal tumor, mesothelioma, epidermoid carcinoma,as well as any other cancers that form solid tumors.

Viruses

Viruses that can be used in the methods of the invention can be derivedfrom any of the members of the family Herpesviridae. For example, herpessimplex virus-1 (HSV-1)-derived viruses can be used. Additional examplesof herpes family viruses from which viruses that are used in theinvention can be derived are herpes simplex virus-2 (HSV-2), vesicularstomatitis virus (VSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV),human herpes virus-6 (HHV-6), human herpes virus-7 (HHV-7), and humanherpes virus-8 (HHV-8). A central feature of the viruses that can beused in the methods of the invention is that they arereplication-competent, and thus are able to infect, replicate in, andlyse malignant cells, while at the same time they are sufficientlyattenuated to not adversely affect normal cells.

Two specific examples of HSV-1-derived viruses that can be used in themethods of the invention are NV1023 (Wong et al., Hum. Gene Ther.12:253-265, 2001) and NV1020, which are described in further detailbelow. An additional specific example of an HSV-1-derived virus that canbe used in the invention is G207 (Yazaki et al., Cancer Res.55(21):4752-4756, 1995). This virus has deletions in both copies of theγ34.5 gene, as well as an inactivating insertion in UL39, which is thegene that encodes infected-cell protein 6 (ICP6), the large subunit ofHSV ribonucleotide reductase.

Still a further specific example of a herpes virus that can be used inthe invention is G47Δ(Todo et al., Proc. Natl. Acad. Sci. U.S.A.98(11):6396-6401, 2001), which is a multimutated, replication-competentHSV-1 vector that was derived from G207 by a 312 basepair deletionwithin the non-essential α47 gene (Mavromara-Nazos et al., J. Virol.60:807-812, 1986). Because of the overlapping transcripts encoding ICP47and US11 in HSV, the deletion in α47 places the late US11 gene undercontrol of the immediate-early α47 promoter, which enhances the growthproperties of γ34.5⁻ mutants.

Additional examples of attenuated HSV viruses that can be used in themethods of the invention include hrR3, which is ribonucleotidereductase-defective (Spear et al., Cancer Gene Ther. 7(7):1051-1059,2000), HF (ATCC VR-260), Maclntyre (ATCC VR-539), MP (ATCC VR-735),HSV-2 strains G (ATCC VR-724) and MS (ATCC VR-540), as well as anyviruses having mutations (e.g., inactivating mutations, deletions, orinsertions) in any one or more of the following genes: the immediateearly genes ICP0, ICP22, and ICP47 (U.S. Pat. No. 5,658,724); the Δ34.5gene; the ribonucleotide reductase gene; and the VP16 gene (i.e., Vmw65,WO 91/02788, WO 96/04395, and WO 96/04394). The vectors described inU.S. Pat. Nos. 6,106,826 and 6,139,834, as well as otherreplication-competent, attenuated herpes viruses, can also be used inthe methods of the invention.

The effects of the viruses used in the methods of the invention can beaugmented, if desired, by including heterologous nucleic acid sequencesencoding one or more therapeutic products in the viruses. For example,nucleic acid sequences encoding cytotoxins, immunomodulatory proteins(i.e., proteins that enhance or suppress patient immune responses toantigens), tumor antigens, antisense RNA molecules, or ribozymes can beincluded in the viruses. Examples of immunomodulatory proteins that canbe encoded by the heterologous nucleic acid sequences include, e.g.,cytokines (e.g., interleukins, for example, any of interleukins 1-15, α,β, or γ-interferons, tumor necrosis factor (TNF), granulocyte macrophagecolony stimulating factor (GM-CSF), macrophage colony stimulating factor(M-CSF), and granulocyte colony stimulating factor (G-CSF)), chemokines(e.g., neutrophil activating protein (NAP), macrophage chemoattractantand activating factor (MCAF), RANTES, and macrophage inflammatorypeptides MIP-1a and MIP-1b), complement components and their receptors,immune system accessory molecules (e.g., B7.1 and B7.2), adhesionmolecules (e.g., ICAM-1, 2, and 3), and adhesion receptor molecules.Appropriate heterologous nucleic acid sequences for use in the methodsof the invention can be readily selected by those of skill in this art.

The heterologous nucleic acid sequences can be inserted into the virusesfor use in the methods of the invention in a location that renders themunder the control of regulatory sequences of the viruses. Alternatively,the heterologous nucleic acid sequences can be inserted as part of anexpression cassette that includes regulatory elements, such as promotersor enhancers. Appropriate regulatory elements can be selected by thoseof skill in the art based on, for example, the desiredtissue-specificity and level of expression. For example, a cell-typespecific or tumor-specific promoter can be used to limit expression of agene product to a specific cell type. This is particularly useful, forexample, when a cytotoxic, immunomodulatory, or tumor antigenic geneproduct is being produced in a tumor cell in order to facilitate itsdestruction, and provides a further safeguard of specificity. Inaddition to using tissue-specific promoters, local (i.e.,intra-resection site) administration of the viruses of the invention canresult in localized expression and effect.

Tumor specific promoters can also be selected for use in the invention,based on the etiology of the cancer. Examples of promoters that functionspecifically in tumor cells include the stromelysin 3 promoter, which isspecific for breast cancer cells (Basset et al., Nature 348:699, 1990);the surfactant protein A promoter, which is specific for non-small celllung cancer cells (Smith et al., Hum. Gene Ther. 5:29-35, 1994); thesecretory leukoprotease inhibitor (SLPI) promoter, which is specific forSLPI-expressing carcinomas (Garver et al., Gene Ther. 1:46-50, 1994);the tyrosinase promoter, which is specific for melanoma cells (Vile etal., Gene Therapy 1:307, 1994; WO 94/16557; WO 93/GB1730); the epidermalgrowth factor receptor promoter, which is specific for squamous cellcarcinoma, glioma, and breast tumor cells (Ishii et al., Proc. Natl.Acad. Sci. U.S.A. 90:282, 1993); the mucin-like glycoprotein (DF3, MUC1)promoter, which is specific for breast carcinoma cells (Abe et al.,Proc. Natl. Acad. Sci. U.S.A. 90:282, 1993); the mts1 promoter, which isspecific for metastatic tumors (Tulchinsky et al., Proc. Natl. Acad.Sci. U.S.A. 89:9146, 1992); the NSE and somatostatin receptor promoters,which are specific for small-cell lung cancer cells (Forss-Petter etal., Neuron 5:187, 1990; Bombardieri et al., Eur. J. Cancer 31A:184,1995; Koh et al., Int. J. Cancer 60:843, 1995); the c-erbB-2 promoter,which is specific for pancreatic, breast, gastric, ovarian, andnon-small cell lung cells (Harris et al., Gene Ther. 1:170, 1994); thec-erbB-3 promoter, which is specific for breast cancer cells (Quin etal., Histopathology 25:247, 1994); and the c-erbB4 promoter, which isspecific for breast and gastric cancer cells (Rajkumar et al., BreastCancer Res. Trends 29:3, 1994). Examples of non-tissue specificpromoters that can be used in the invention include the earlyCytomegalovirus (CMV) promoter (U.S. Pat. No. 4,168,062) and the RousSarcoma Virus promoter (Norton et al., Mol. Cell Biol. 5:281, 1985).Also, HSV promoters, such as HSV-1 IE and IE 4/5 promoters, can be used.

Any of a number of well-known formulations for introducing viruses intocells in patients can be used in the invention. (See, e.g., Remington'sPharmaceutical Sciences (18^(th) edition), ed. A. Gennaro, 1990, MackPublishing Co., Easton, Pa.) However, the viruses can be simply dilutedin a physiologically acceptable solution, such as sterile saline orsterile buffered saline, with or without an adjuvant or carrier. Theamount of virus to be administered can readily be determined by those ofskill in this art, and depends on factors such as, for example, thecondition of the patient intended for administration (e.g., the weight,age, and general health of the patient), the mode of administration, andthe type of formulation. In general, an effective dose of, e.g., fromabout 10¹ to 10¹⁰ plaque forming units (pfu), for example, from about5×10⁴ to 1×10⁶ pfu, e.g., from about 1×10⁵ to about 4×10⁵ pfu, isadministered, although the most effective ranges may vary from patientto patient, as can readily be detennined by those of skill in this art.

The viruses are administered to sites of surgical resection in patientsby, for example, injection directly into the surgical bed afterresection of a primary tumor, either before or after closing of thesurgical site. Alternatively, as is discussed above, the viruses can beinjected directly into tumors.

The methods of the invention can employ replication competent,attenuated herpes viruses as sole therapeutic agents or, alternatively,these agents can be used in combination with other anticancertreatments. Examples of additional therapies that can be used includechemotherapy, biological therapy, gene therapy, radiation therapy,antisense therapy, and therapy involving the use of angiogenesisinhibitors (e.g., angiostatin, endostatin, and icon). Selection of anyof these types of therapies for use with replication-competent,attenuated herpes in the methods of the invention can readily be carriedout by those of skill in the art.

Specific examples of chemotherapeutic agents that can be used in themethods of the invention are provided as follows. These compounds fallinto several different categories, including, for example, alkylatingagents, antineoplastic antibiotics, antimetabolites, and natural sourcederivatives. Examples of alkylating agents that can be used in themethods of the invention include busulfan, carboplatin, carnustine,chlorambucil, cisplatin, cyclophosphamide (i.e., cytoxan), dacarbazine,ifosfamide, lomustine, mecholarethamine, melphalan, procarbazine,streptozocin, and thiotepa; examples of antineoplastic antibioticsinclude bleomycin, dactinomycin, daunorubicin, doxorubicin, idarubicin,mitomycin (e.g., mitomycin C), mitoxantrone, pentostatin, andplicamycin; examples of antimetabolites include fluorodeoxyuridine,cladribine, cytarabine, floxuridine, fludarabine, flurouracil (e.g.,5-fluorouracil (5FU)), gemcitabine, hydroxyurea, mercaptopurine,methotrexate, and thioguanine; and examples of natural sourcederivatives include docetaxel, etoposide, irinotecan, paclitaxel,teniposide, topotecan, vinblastine, vincristine, vinorelbine, taxol,prednisone, tamoxifen, asparaginase, and mitotane.

The biological therapy that can be used in the methods of the inventioncan involve administration of an immunomodulatory molecule, such as amolecule selected from the group consisting of tumor antigens,antibodies, cytokines (e.g., interleukins, interferons, tumor necrosisfactor (TNF), granulocyte macrophage colony stimulating factor (GM-CSF),macrophage colony stimulating factor (M-CSF), and granulocyte colonystimulating factor (G-CSF)), chemokines, complement components,complement component receptors, immune system accessory molecules,adhesion molecules, and adhesion molecule receptors.

The methods of the invention, as described herein, are based, in part,on the experimental results that are described as follows.

EXPERIMENTAL RESULTS

Summary

Oncolytic herpes viruses have significant antitumor effects in animalmodels when delivered directly to established tumors. Lymphaticmetastases are a common occurrence for many tumor types. This studyinvestigates the use of an attenuated, replication-competent, oncolyticherpes simplex virus (NV1023), both to treat a primary tumor by directinjection, and to travel through the lymphatic system to treatmetastatic tumors within the lymph nodes draining lymph from the site ofprimary cancer. Isosulfan blue dye was injected into murine auricles todetermine normal lymphatic drainage patterns, and demonstratedconsistent blue staining of a group of ipsilateral cervical lymph nodes.Auricular injections of NV1023 resulted in viral transit to these lymphnodes, as measured by X-gal histochemistry and viral plaque assay. Usingthe SCC VII cell line, a novel murine model of auricular squamous cellcarcinoma was developed with an approximately 20% incidence of cervicallymph node metastases. Delivery of NV1023 to surgical beds followingexcision of auricular SCC VII tumors resulted in successful viralinfection of metastatic SCC VII cells within the cervical lymph nodes.After a 7 week follow-up, significantly enhanced locoregional control(p<0.05, Fischer's exact test) and disease free survival (p<0.05, Logrank test) were evident with NV1023 treatment. This study demonstratesthat the delivery of NV1023 to a primary tumor site following surgicalexcision reduces both primary site recurrence and regional nodalmetastases.

Lymph Node Drainage Patterns

Isosulfan blue dye (100 μl) was injected into the base of the leftposterior auricle to identify the normal draining lymph nodes for thisanatomic site. In all cases (n=5), intense blue dye was visible within agroup of 1-3 ipsilateral cervical lymph nodes adjacent to the externaljugular vein and salivary gland tissue. These cervical lymph nodes wereconsistently identified as the primary draining nodes to the auricularregion (FIGS. 1A and 1B). Contralateral cervical lymph nodes andipsilateral nodes deep to the sternocleidomastoid muscle did not stainblue in any animal.

SCC VII Auricular-Cervical Metastatic Model

To develop a model of cervical lymphatic metastases, SCC VII tumors wereimplanted in the left auricles of mice and grown to 13-18 mm in largestdimension to allow for the microscopic seeding of cervical lymph nodes.The growth of these auricular tumors did not cause significantmorbidity, and did not impair either feeding or respiration. Theauricular tumors were then excised to control primary site morbidity, toprolong survival, and to permit the subsequent development of palpablecervical node metastases.

The implantation and excision of auricular SCC VII tumors in C3H/HeJmice led to approximately 20% of these animals developing palpableadenopathy in the ipsilateral neck within the following two weeks (FIGS.1C and 1D). Histologic examination confirmed the presence of metastaticsquamous cell carcinoma in cases of palpable nodes, which weregenerally >8 mm in dimension. Histologic examination of excised lymphnodes demonstrated that metastatic SCC VII cells are deposited in thesubcapsular sinus of the lymph node before progressively infiltratingthe nodal parenchyma and replacing the entire nodal architecture (FIGS.2A and 2B). Primary site recurrence at the sites of primary auriculartumor resection was noted in approximately 10% of cases.

Viral Transit From Auricle to Cervical Lymph Nodes

The ability of an oncolytic virus to travel from the auricle to thedraining cervical lymph nodes was demonstrated by injecting NV1023 intothe left auricle of non-tumor bearing animals and histologicallyexamining the ipsilateral draining lymph nodes at 24 and 48 hours forX-gal staining cells. At 24 hours, there was positive X-gal stainingwithin the ipsilateral cervical lymph nodes (FIG. 3A). Blue stainedcells tended to be sparse and scattered. At 48 hours, most ipsilateraldraining nodes were negative for blue cells. Contralateral lymph nodesat both 24 and 48 hours were negative for X-gal staining cells.

Successful viral transit from auricle to the cervical lymph nodes wasfurther confirmed by using the GFP-expressing NV1066 virus. NV1066 wasinjected into the left posterior auricle and the cervical lymph nodeswere harvested 24 hours later. Fluorescent microscopy of ipsilateralcervical lymph nodes demonstrated the presence of sparse, scatteredgreen fluorescence, reflecting the presence of NV1066-infected cells(FIGS. 3B and 3C).

The number of recoverable viral plaque forming units (pfu) from thedraining lymph nodes was also determined by viral plaque assay. Draininglymph nodes excised 10 minutes after auricular viral injections ofNV1023 yielded approximately 5000 viral pfu/gm of nodal tissue. No livevirus was recovered from any ipsilateral nodes excised 24 hours afterauricular viral injection, or from any contralateral lymph nodes excisedat either 10 minutes or 24 hours. This transient and sparse appearanceof virus within the lymphatics of animals not bearing cancer is as wouldbe expected from viruses designed to have limited infectivity fornon-cancerous tissues.

Viral Therapy of SCC VII Auricular Tumors

To determine the in vivo efficacy of NV1023 against established SCC VIItumors, NV1023 was injected as three serial doses into establishedauricular tumors and subsequent tumor dimensions recorded. Average tumorvolumes for the NV1023 treated animals were significantly decreased ascompared to controls (p<0.0001 at day 7, t-test, FIG. 4).

Viral Therapy of SCC VII Cervical Metastases

NV1023 was delivered to the surgical bed after excision of establishedauricular SCC VII tumors. At 24 hours after viral delivery, animalsunderwent neck exploration, cervical node excision, and histologicexamination of bilateral nodal groups. X-gal staining revealed thepresence of blue-staining metastatic SCC VII deposits within the lymphnodes (FIGS. 5A and 5B). X-gal staining was minimal in adjacent normallymphocytes and in lymph nodes without metastatic SCC VII cells.

A survival experiment was performed by comparing surgical bed treatmentwith either PBS (n=28) or NV1023 (n=28) following the excision ofauricular tumors. Animals were subsequently monitored for either primary(auricular) recurrence or the development of regional (cervical)metastases. The average cervical nodal volume of the PBS treated group(440 mm³) was greater than that of the NV1023 treated group (98 mm³) atday 15 (FIG. 6). Of the 28 animals receiving PBS, 3 (10.7%) developedprimary site recurrences at the auricular excision site, and 5 (17.9%)developed palpable nodal metastases in the ipsilateral neck, for a totalof 8 (28.6%) locoregional failures. Of the 28 animals receiving NV1023,1 (3.6%) developed a primary site recurrence and 1 (3.6%) developed apalpable nodal metastasis, for a total of 2 (7.1%) locoregionalfailures. There were no cases of both primary site recurrence and nodalmetastasis occurring within the same animal. There was also no evidenceof distant metastases in either group. The NV1023-treated group showed asignificantly enhanced locoregional control rate (p<0.05, Fischer'sexact test) as compared to the PBS-treated control group. With afollow-up period of 7 weeks, disease free survival was significantlyenhanced (p<0.05, Log rank test) for the NV1023-treated group (FIG. 7).

There was also no evidence of any morbidity resulting from NV1023administration. There was no significant weight loss, mucosal orcutaneous ulcerations, neurotoxicity, or other toxicities detected inany of the virally treated animals. All auricular incision sitesdemonstrated rapid and complete wound healing following NV1023administration to the surgical bed.

MATERIALS AND METHODS

Cell Lines

The murine SCC VII cell line is a cutaneous squamous cell carcinoma thatspontaneously arose from the C3H/HeJ mouse. SCC VII (H. Suit, HarvardUniversity) is a rapidly dividing cell line with an estimated doublingtime of 18 hours (Fu et al., Int. J. Radiat. Oncol. Biol. Phys.10:1473-1478, 1984; O'Malley et al., Arch. Otolaryngol. Head Neck Surg.123:20-24, 1997). SCC VII cells were grown in vitro in MEM containing10% FCS at standard cell culture conditions. African green monkey kidney(Vero) cells for viral plaque assays were also grown in MEM containing10% FCS at standard cell culture conditions (American Type CultureCollection, Manassas, Va.).

Viruses

NV1023 is an attenuated, replication-competent, oncolytic herpes viruswhose construction has been previously described in detail (Wong et al.,Hum. Gene Ther. 12:253-265, 2001). NV1023 carries a non-functional, 5.2kb fragment of HSV-2 DNA in the U_(L/S) junction. This HSV-2 fragmentwas originally inserted into the NV1020 (R7020) virus, from which NV1023was derived, to broaden its potential application as a herpes vaccine(Meignier et al., J. Infect. Dis. 158:602-614, 1988). NV1023 isattenuated by a 15 kilobase deletion in the inverted repeat U_(L/S)junction that deletes one copy of the γ₁134.5 neurovirulence gene andthe UL56 gene. NV1023 also contains the E. coliβ-galactosidase (lacZ)gene inserted at the US10-12 locus to serve as a marker of infection.

NV1020 (Medigene Inc., San Diego, Calif.) is an attenuated,replication-competent derivative of herpes simplex virus type-1 (HSV-1)(Delman et al., Hum. Gene Ther. 11:2465-2472, 2000). NV1020 is anon-selected clonal derivative from R7020, a candidate HSV-1/2 vaccinestrain that was obtained from Dr. B. Roizman (Meigner et al., J. Infect.Dis. 158:602-614, 1998). The structure of NV1020 is characterized by a15 kilobase deletion encompassing the internal repeat region, leavingonly one copy of the following genes, which are normally diploid in theHSV-1 genome: ICP0, ICP4, the latency associated transcripts (LATs), andthe neurovirulence gene γ₁34.5. A fragment of HSV-2 DNA encoding severalglycoprotein genes was inserted into this deleted region. In addition, a700 basepair deletion encompasses the endogenous thymidine kinase (TK)locus, which also prevents the expression of the overlapping transcriptsof the U_(L)24 gene. An exogenous copy of the HSV-1 TK gene was insertedunder control of the α4 promotor. Virus was propagated in Vero cells andharvested by freeze thaw lysis to release virus from the cell fraction.Cell lysates were clarified by centrifugation, and viral titers weredetermined on Vero cells by plaque assay. All virus preparations wereformulated in D-PBS-10% glycerin and stored at −80° C.

Animals

All animal procedures were approved by the Memorial Sloan-KetteringInstitutional Animal Care and Use Committee. Six-week old male C3H/HeJmice (Jackson Laboratory, Bar Harbor, Me.) were anesthetized withinhalational methoxyflurane for injections of isosulfan blue dye, SCCVII tumor cells, and NV1023 or NV1066 virus. Each animal received anintraperitoneal injection of ketamine (70 μg) and xylazine (20 μg) in100 μl of sterile water prior to the surgical excision of auriculartumors. Animals were sacrificed by CO₂ inhalation.

Lymph Node Drainage Patterns

The normal lymphatic drainage pattern of the auricular region wasdetermined by injecting 1% isosulfan blue dye (100 μl) into the base ofthe posterior left auricle of C3H/HeJ mice (n=5). At two minutesfollowing injection, mice were sacrificed, their necks surgicallyexplored, and the draining cervical nodes visually identified by thepresence of blue dye.

Development of SCC VII Auricular-Cervical Metastatic Model

A novel head and neck metastatic model of murine squamous cell carcinomawas developed. Auricular tumors were established by the injection of1×10⁶ SCC VII cells in 50 μl PBS into the base of the left posteriorauricle of each mouse. By day 13, the auricular tumors ranged from 13-18mm in greatest dimension. All tumors were then completely surgicallyexcised with the left auricle, and the incision was closed with arunning 4-0 nylon suture.

Over a two to three week postoperative period, animals were monitoredfor the subsequent development of palpable adenopathy in the ipsilateralneck. At varying time points following tumor excision, animals weresacrificed and their necks surgically explored. Enlarged cervical nodeswere excised, immediately frozen in imbedding media (Tissue Tek, SaguraInc., Torrance, Calif.), cut into 6 μm thick sections, stained withhematoxylin and eosin, and examined histologically to identify thepresence of metastatic squamous cell carcinoma.

Viral Transit From Auricle to Cervical Lymph Nodes

To document the ability of virus to travel from the auricle to thecervical lymph nodes, NV1066 or NV1023 was injected at a dose of 2×10⁷pfU/100 μl of phosphate buffered saline (PBS) into the base of the leftposterior auricle in non-cancer-bearing C3H/HeJ mice. After 24 or 48hours, mice were sacrificed and their necks surgically explored.Ipsilateral and contralateral cervical lymph nodes were excised, frozenin Tissue Tek, cut into 6 μm thick sections, mounted on glass slides,washed in PBS, and examined.

Nodes from animals injected with NV1066 and from control animals wereexamined under fluorescence microscopy at wavelengths from 515-585 nm,and GFP expression identified by the presence of fluorescent greencolor. Sections were also stained with 20 μl of 4,6-diamino-2phenylindole (DAPI, 0.1 μg/ml) in mounting media (1 mgp-phenylenedamine/1 cc of 80% glycerol in PBS) to identify cellularnuclei by blue fluorescence.

Nodes from animals injected with NV1023 and from controls were stainedwith 5-bromo-4-chloro-3-indol-β-D-galactopyranoside (X-gal) at 37° C.for 2 hours, as previously described (Geller et al., Science241:1667-1669, 1988) for assessment of β-gal expression. Counterstainingof background cell nuclei with nuclear fast red was performed. Virallyinfected cells expressing β-Sgalactosidase were identifiedhistologically as blue-staining cells.

To measure viral recovery from the cervical lymph nodes, NV1023 wasagain injected at a dose of 2×10⁷ pfU in 100 μl of PBS into the leftauricles of mice. At 10 minutes (n=3) and 24 hours (n=3) following viralinjection, animals were sacrificed and the bilateral cervical lymphnodes were surgically excised, weighed, homogenized in 250 μl of PBS,mixed, and subjected to three freeze-thaw cycles to lyse cells. After asecond centrifugation (30 seconds, 10,000 rpm), supernatants werecollected and titered on confluent Vero cells, as previously described,to determine the quantity of viral plaque forming units recovered (Wonget al., Hum. Gene Ther. 12:253-265, 2001).

Viral Therapy of SCC VII Auricular Tumors

Auricular tumors were established by the injection of 5×10⁵ SCC VIIcells in 50 μl PBS into the base of the left posterior auricle inC3H/HeJ mice. Visible tumors developed in all animals within 34 days. Byday 6, tumors were approximately 5-6 mm in greatest dimension, andanimals were distributed into two groups of equitable tumor volumes. Onegroup (n=8) was treated with three serial intratumoral injections ofNV1023 at 2×10⁷ pfu in 100 μl PBS (delivered every other day). The othergroup (n=8) received an identical regimen of PBS injections as acontrol. Subsequent tumor dimensions were recorded and volumescalculated by the formula for the volume of an ellipsoid:volume=(4/3)*π*(length/2)*(width/2)².

Viral Therapy of SCC VII Cervical Metastases by Injection of PrimaryTumor Sites

Auricular SCC VII tumors were established as described above. On day 13after tumor cell injection, tumor volumes were measured and animals weredivided into two groups with equitable tumor volumes. Auricular tumorswere completely excised in all mice. Immediately after tumor excisionand wound closure with 4-0 nylon suture, one group of animals (n=28) wastreated with NV1023 and the other group (n=28) with PBS. NV1023 at adose of 5×10⁷ pfu in 100 μl PBS was injected through the closed incisionline and into the potential space between the skin and the surgical bed.The control group of animals underwent identical injections of 100 μlPBS. A separate group of animals (n=10) was treated identically withNV1023, and cervical lymph nodes were subsequently excised 24 and 48hours later and examined by histochemical staining for β-galactosidaseexpression.

Animals were routinely weighed and monitored postoperatively for thedevelopment of palpable cervical metastatic disease, primary site(auricular) recurrence, or any toxicity related to tumor growth or virusadministration. The dimensions of any palpable cervical adenopathy thatsubsequently developed were measured with calipers, and nodal volumescalculated. Animals were sacrificed if the greatest nodal dimension orprimary site recurrence exceeded 18 mm, if there was evidence of skinulceration, or if there was any other morbidity evident.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindependent publication or patent application was specifically andindividually indicated to be incorporated by reference.

1. A method of preventing or treating cancer in a subject, said methodcomprising the steps of: surgically resecting a tumor from the subject;and administering an attenuated, replication-competent, oncolytic herpesvirus to the site of surgical resection.
 2. The method of claim 1,wherein said cancer is present at the site of surgical resection.
 3. Themethod of claim 1, wherein said cancer has metastasized from the site ofsurgical resection.
 4. The method of claim 3, wherein said cancer ispresent in the lymphatic system of said subject.
 5. The method of claim4, wherein said cancer is present in a lymph node of said subject. 6.The method of claim 1, wherein said herpes virus is a herpessimplex-1-derived virus.
 7. The method of claim 6, wherein said herpesvirus is NV1023.
 8. The method of claim 1, wherein said subject is ahuman.
 9. The method of claim 1, wherein said herpes virus isadministered to said subject by injection.
 10. The method of claim 1,wherein said herpes virus comprises a heterologous nucleic acid moleculeencoding a therapeutic product.
 11. The method of 10, wherein saidtherapeutic product is selected from the group consisting of cytotoxins,immunomodulatory proteins, tumor antigens, antisense nucleic acidmolecules, and ribozymes.
 12. The method of claim 1, further comprisingadministering a second anticancer treatment to said subject.
 13. Themethod of claim 12, wherein said second anticancer treatment is selectedfrom the group consisting of chemotherapy, biological therapy, radiationtherapy, and gene therapy.
 14. The method of claim 1, further comprisinginjecting an attenuated, replication-competent, oncolytic herpes virusinto said tumor of said subject prior to exision. 15-21. (canceled) 22.The method of claim 14, further comprising administering a secondanticancer treatment to said subject.
 23. The method of claim 22,wherein said second anticancer treatment is selected from the groupconsisting of chemotherapy, biological therapy, radiation therapy, andgene therapy. 24-27. (canceled)